Bacterial type III effector proteins are the primary virulence factors that guide the progression of numerous Gram-negative bacterial infectious diseases. Recent studies have estimated that a single pathogen delivers 10-50 unique effector proteins directly into host cells. Collectively, these virulence factors hijack host innate immune response and facilitate bacterial replication, dissemination, and disease progression. Therefore, determining how bacterial effector proteins control host intracellular communication pathways at the structural, biochemical, and biophysical level is an ongoing challenge in infectious disease research. This proposal seeks to reveal a structural and functional understanding of these host-pathogen relationships. Prior to this proposal, we identified a class of Enterohaemorhagic E. coli effector proteins that directly regulates host membrane trafficking GTPases and a cell polarity kinases through unique structural interactions. Using these structures as a guide, we will determine the molecular mechanism for bacterial regulation of human signaling enzymes. This includes determining how bacteria hijack host cargo trafficking pathways by directly regulating ARF GTPase activity on a membrane surface (Aim 1). We will also examine a novel PAK kinase activation mechanism through a series of X-ray crystallography, small molecule inhibitor studies, and enzymatic assays (Aim 2). The resulting structure-based theories will be used to directly compare the actions of numerous bacterial type III effector homologs to assemble host enzymes into new signaling circuits on the surface of bacterial effector scaffolds (Aim 3). Developing new drugs that target bacterial effector - host enzyme complexes would be an innovative approach to combat emerging infectious disease. While this idea holds great potential, the paucity of mechanistic information gleaned from virulence factor structures has so far hampered their development as suitable drug targets. As a means to this end, these studies will allow us to predict new mechanisms of action for understudied bacterial effector proteins, and provide a glimpse into the structural-based evolutionary progression of a related pathogen group.